Assessing the Highest Level of Evidence from Randomized Controlled Trials in Omega-3 Research

Over the years, there has been heightened interest in the health benefits of n-3 polyunsaturated fatty acids (PUFA) in reducing chronic diseases such as, cardiovascular disease (CVD), cancer, type 2 diabetes, and acute macular degeneration (AMD). Due to inconsistent findings in the evidence, a review to critically examine the plethora of evidence from randomized controlled trials (RCTs) in n-3 PUFA research was undertaken. The aim of this review is to study the highest level of evidence and to identify gaps in n-3 PUFA research. RCTs were originally designed for pharmaceutical research and later adopted for nutrition and food-related research. RCTs with active diseases assume that n-3 PUFA will have “drug” like effects, and this high expectation may have led to the inconsistent evidence in the literature. The inconsistency in the literature may be related to varying doses of n-3 PUFA, sources of n-3 PUFA (food vs. supplement; plant vs. marine), type of n-3 PUFA (mixture vs. purified), trial duration, population characteristics, sample size, and genetic variation. For future research, there is a need to distinguish between primary and secondary prevention, and to focus RCTs on primary prevention of chronic diseases by n-3 PUFA which is lacking in the literature.


Introduction
N-3 long chain polyunsaturated fatty acids (n-3 PUFA) have an important role in human health and reduction in chronic diseases [1]. N-3 PUFA are presumed to have antiinflammatory and anti-thrombotic properties, lower plasma triglycerides and low-density lipoprotein (LDL) cholesterol, improve vasomotor and endothelial functions, and inhibit cell growth factors [2,3]. There are three main types of n-3 PUFA, and they are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). EPA and DHA most specifically are vital for improved cardiovascular functions, neurodevelopment, and in improving metabolic and immune processes [1]. However, it is challenging to obtain enough EPA and DHA solely from diet as they cannot be synthesized by the body and must therefore be supplemented through fish and fish oil supplements [4]. Since EPA and DHA are important for human health, numerous health organizations have recommended 250-500 mg of EPA and DHA per day [1]. While the importance of EPA and DHA for human health is recognized, there are still no established dietary reference intakes (DRIs) for EPA and DHA, though some researchers have been calling for it as this may help "inform nutrition policy decisions and reduce consumer uncertainty" [5].
Over the years, due to industrialization, there has been a reduction in the intake of n-3 PUFA, and increased consumption of highly processed foods rich in saturated fats and n-6 PUFA [6]. Consequently, a diet low in n-3 PUFA and high in saturated fats and n-6 PUFA is associated with poorer metabolic and cardiovascular health in both men and women [6]. Because of heightened interest in the health benefits of n-3 PUFA, there has been an increased intake of fish oil supplements among the general public, and a plethora with an Initial Glargine Intervention (ORIGIN) [25] and a Study of Cardiovascular Events in Diabetes (ASCEND) [29] studied the effects of n-3 PUFA on cardiovascular outcomes in patients with impaired fasting glucose or with diabetes, but without evidence of atherosclerotic CVD. Both had more than 12,000 participants enrolled, taking 0.84 g of DHA + EPA for more than 6 years, and still failed to show any significant reduction in CVD and its related outcomes [25,29]. Moreover, the Vitamin D and Omega-3 Trial (VITAL), a study of 25,871 subjects with no previous CVD risk factors taking 0.84 g EPA + DHA with 2000 IU of vitamin D daily for more than 5 years also reported no significant reduction in major cardiovascular events or mortality [30]. Likewise, two other large-scale trials, namely the Long-Term Outcomes Study to Assess Statin Residual Risk with Epanova in High Cardiovascular Risk Patients with Hypertriglyceridemia (STRENGTH) and the Omega-3 fatty acids in Elderly with Myocardial Infarction (OMEMI) demonstrated null findings after combined EPA + DHA therapy [31,32]. The STRENGTH trial was even stopped early because of a low possibility of demonstrating any clinical benefits [31].

Cancer
In 2020 alone, cancer was the leading cause of morbidity and death globally with approximately 19.3 million new cancer cases and 10 million cancer-related deaths [33]. Since cancer incidence and mortality are on the rise, it has been suggested that the intake of n-3 PUFA may reduce cancer risk by regulating metabolic pathways and inflammatory responses, oxidative stress, and changes in the composition of membrane affecting cell signaling pathways [34,35]. It was demonstrated in vitro and in animal studies that n-3 PUFA may inhibit breast cancer growth [36]. Asian countries with the highest intake of n-3 PUFA had a significantly lower rate of breast cancer compared to Europe and United States [37]. A greater incidence of breast cancer was further observed among Asian-American women migrating to the West due to a shift from their traditional fish-based diet to a high-fat Western diet [38]. Earlier evidence focusing on Asian populations showed a protective benefit of n-3 PUFA, with a 20% and 26% reduction in breast cancer risk in both Japanese and Singaporean Chinese populations, respectively [39,40]. A more recent dose-response meta-analysis demonstrated a 6% decrease in breast cancer risk for every 1% increase in circulating n-3 PUFA [41].
However, the evidence of n-3 PUFA on cancer development and cancer mortality risk remains contradictory when considering all cancer types. Epidemiological studies demonstrating significant reductions in cancer risk may have overestimated the effects of n-3 PUFA supplementation, leading us to believe that estimates of cancer risk need to be assessed by cancer type and it is not possible to generalize across all cancers. An earlier SRMA of observational studies showed significant associations between n-3 PUFA and cancer incidence, especially colorectal, lung, prostate, and breast cancer [42]. More than three decades ago, the Lyon Diet Heart Study, a trial consisting of 605 MI survivors, reported that supplementing the Mediterranean diet with a margarine rich in n-3 PUFA reduced cancer risk by 61% after a 4 year follow up [43]. In contrast, a current SRMA of RCTs extensively examining the high-quality evidence of the effects of n-3 PUFA on cancer demonstrated little or no effect on cancer diagnosis and cancer mortality [12]. Consequently, the same study reported a slight increase in prostate cancer risk after an intake of ALA, although the certainty of evidence was of low-quality [12]. Most recently, large-scale RCTs such as GISSI-P, JELIS, ORIGIN, ASCEND, and VITAL showed null effects on cancer incidence and cancer mortality after more than 3.5 years of follow-up and with doses of EPA + DHA combined ranging from 0.84-1.8 g [18,29,30,[44][45][46] (Table 2). Some small scale RCTs also examining the effects of n-3 PUFA on cancer incidence and mortality for a mean follow-up of 1.5 years found no association with cancer outcomes [47][48][49] (Table 2).

Type 2 Diabetes
With more than 700 million people projected to be diagnosed with type 2 diabetes by 2045, there is a need to prevent and manage this endocrine disease because of its association with increased risks of CVD and cancer [50]. Type 2 diabetes is also associated with a variety of modifiable risk factors that can be prevented by following a healthy diet and lifestyle [51]. Thus, people are now turning to n-3 PUFA supplementation to manage the symptoms associated with type 2 diabetes such as elevated blood glucose and insulin resistance [52]. Over the years, animal and cell culture studies have shown that n-3 PUFA may prevent type 2 diabetes through anti-inflammatory properties, insulin signaling, changing cell membrane function and controlling expression of glucose metabolism genes [53]. For the past decade, there has been an influx of studies investigating the effects of n-3 PUFA on the prevention and management of type 2 diabetes [52]. But as observed in the case of CVD and cancer prevention and treatment, the evidence for a protective effect of n-3 PUFA on type 2 diabetes and its related parameters also remains inconsistent [51,52].
A SRMA of 12 RCTs studying the effects of n-3 PUFA on glucose control reported no reduction in fasting insulin (FINS), glycosylated hemoglobin (HbA1c), and HOMA of insulin resistance (HOMA-IR) levels [51]. Another SRMA of 25 RCTs investigating the effects of n-3 PUFA supplementation on type 2 diabetes treatment or prevention showed a significant reduction in fasting blood glucose and insulin resistance, but no significant effect on HbA1c was observed [52]. Trials examining the effects of n-3 PUFA on type 2 diabetes among those with impaired glucose tolerance or with metabolic syndrome also found no significant changes in HbA1c, fasting glucose, and insulin sensitivity with the doses of n-3 PUFA ranging from 0.6 to 6 g per day for a duration ranging from 8 weeks to 1 year [54][55][56][57][58] (Table 3). Conversely, other trials reported a significant reduction in fasting blood glucose, insulin resistance, and HbA1c after supplementing the diet with dosage of n-3 PUFA ranging from 0.41 to 3 g/day for a follow-up of 8 weeks to 1 year as well [59][60][61][62][63] (Table 3). One particular RCT conducted among patients with vitamin D deficiency reported a significant increase in HbA1c in the n-3 PUFA group after 8 weeks of supplementation with 0.3 g of n-3 PUFA daily and 50,000 IU of vitamin D weekly [64].

Macular Degeneration
Age-related macular degeneration (AMD) accounts for 8.7% of blindness globally in people 60 years and older, while the number of people with AMD is expected to increase to 288 million by 2040 [71]. Consumption of n-3 PUFA has been postulated as a potentially effective strategy to protect against AMD [72]. Evidence from prospective cohort, crosssectional, and case-control studies has previously shown an association with intake of fatty fish consumption or n-3 PUFA and reduced risk of AMD [73][74][75]. A newly published dose-response meta-analysis of 11 prospective cohorts reported that an increment of 1 g of EPA + DHA daily reduced early AMD risk by 50-60% [76]. Conversely, a review examining high quality evidence from only two RCTs demonstrated that supplementation of n-3 PUFA for up to 5 years did not reduce the incidence or risk of progression to advanced AMD [77].
The Age-Related Eye Disease Study 2 (AREDS2), a large-scale study of 4203 participants with a mean age of 73.1 years and at risk for progression to advanced AMD, reported that 1 g of EPA + DHA combined with lutein and zeaxanthin did not reduce the progression to advanced AMD after a median follow-up of 5 years [78] (Table 4). Another large-scale trial, the VITAL study found no overall effect of n-3 PUFA on AMD incidence or progression among 25,871 participants with a mean age of 67.1 years after a median follow-up of 5.3 years [79] (Table 4).

Conflicting Evidence in RCTs, "The Gold Standard"
In nutrition research, RCTs are considered the "gold standard" as they are known for their robustness, high-quality evidence, and ability to establish causality between exposure and outcomes [80]. Evidence-based nutrition guidelines, public health initiatives, and nutrition health claims are based on cumulative data obtained from systematic reviews and meta-analyses of RCTs [81]. As mentioned by Musa-Veloso et al., RCTs were originally designed to "test the efficacy of pharmaceuticals", and since then, have been "adopted" in nutrition research [81]. But RCTs in nutrition research come with their share of challenges, which are not seen in drug-related research. Recruitment and selection of study participants, methods of randomization, habitual intake of nutrients of interest by study participants, blinding of study personnel and participants, adherence and compliance to the treatment regimen, identification of a suitable comparator, baseline nutrient and health status of study participants, and statistical analyses are among the various challenges faced when conducting nutrition related RCTs [80,81].
RCTs with active diseases assume that n-3 PUFA will have "drug" like effects. However, very few nutrients have "drug" like effects and only under acute deficiency conditions such as scurvy, rickets and osteoporosis. N-3 PUFA maintains its anti-inflammatory, antithrombotic and anti-tumor properties through multiple mechanisms such as immunomodulation, gene expression, and oxylipin synthesis [82,83]. Perhaps there is a need to recognize that even with disease prevention-like properties, n-3 PUFAs act long-term and it is challenging to observe short term health benefits, especially in the context of short term acute RCTs. Therefore, it is not surprising that the general conclusion that while RCTs rigorously measure the impact of an intervention by establishing the cause-effect relationship with less bias, the research surrounding n-3 PUFA, and the aforementioned chronic diseases remains conflicting [84].
In this review, the evidence from RCTs examining effects of n-3 PUFA on CVD, cancer, type 2 diabetes, and AMD, has been thoroughly summarized and the following observations are noted. The significance in reducing CVD incidence and risk by n-3 PUFA studies may depend on whether the trials are investigating primary or secondary prevention; the varying doses of n-3 PUFA and duration across trials; the different characteristics and health status of study populations where some studies included participants with higher cardiovascular risks while others included those with lower CVD risks; the large sample size of some RCTs; the daily intake of n-3 PUFA, supplemental or food form of n-3 PUFA, as well as the genetic variation influencing the absorption of n-3 PUFA [8]. Furthermore, the differences in the design of the trials may contribute to the significance of the results and two major examples are the GISSI-P and JELIS as open-label trials demonstrating significant reduction in CVD outcomes.
In GISSI-P, an open-label trial with a focus on secondary prevention, the significant reduction in total CVD, CVD mortality, and CHD mortality may be because participants who were survivors of their first MI, were also adhering to intensive CVD-lowering regimens such as aspirin, beta-blockers, angiotensin enzyme inhibitors, statins, as well as following a Mediterranean diet [44]. Since the participants were also administered 300 mg of vitamin E daily, it could have potentially prevented LDL oxidation, therefore lowering CVD events [85,86]. Moreover, the intake of cholesterol-lowering medications increased from 5% at baseline to 45.5% at 42 months, which may not make its results generalizable to other populations [44].
Consistent with secondary prevention studies, JELIS and REDUCE-IT are two other large-scale trials demonstrating significant reduction in CVD events [18,20]. The substantial reduction in non-fatal coronary events by 19% in the JELIS trial was mostly attributed to the administration of "highly purified EPA" instead of EPA + DHA or fish oils [18]. The high intake of fish among Japanese as compared to Western populations may have also contributed to the significant reduction in non-fatal coronary events, highlighting country-specific differences [18,87]. Moreover, to accurately determine the fish intake of JELIS population, plasma EPA fatty acid concentrations at baseline were measured and a value of 2.9 mol% was obtained, which was relatively high compared to a value of 0.3 mol% for the average US population [18]. Therefore, the significant results obtained from the JELIS trial cannot be generalized to other populations due to the elevated plasma EPA fatty acid levels among Japanese. Additionally, both treatment groups in JELIS were prescribed a low dose of statin medications as recommended by Japan's Ministry of Health, Labor, and Welfare, known to lower lipid levels and major coronary events [88,89]. However, since 67% of the JELIS participants were females, a lower rate of coronary events was observed as women were 2.3 times less likely than men to have an incidence of coronary events [88]. The biological effects of EPA such as lowering thrombosis risks, inflammation and arrythmia, and reduction in triglycerides may also be postulated to reduce non-fatal coronary events among the JELIS participants [18].
Furthermore, in the REDUCE-IT trial, the numerous mechanisms of n-3 PUFA, as well as the daily intake of 4 g of EPA may have significantly reduced ischemic events [90]. As mentioned by Bhatt et al., the "EPA-related effects" such as "aggregate contribution" may have largely reduced the incidence of total ischemic events in the group receiving EPA alone [90]. Although the population of the trial were statin-treated and had low LDL-C, they also had elevated triglyceride levels and were at high risk for ischemic events, which potentially contributed to the significant reductions in ischemic events in the EPA group [90]. The use of mineral oil versus corn oil as a comparator in the REDUCE-IT trial may have contributed to the positive results of the trial because of its negative effects on Apo-B, LDL-cholesterol, and hs-CRP levels [20,91]. Conversely, large-scale RCTs such as ASCEND, ORIGIN, Risk and Prevention, STRENGTH, and VITAL failed to demonstrate significant reductions in major cardiovascular events after a follow-up ranging from 2-7.4 years and with a dosage range of 0.84-4 g of EPA + DHA [25,26,[29][30][31]. These large-scale trials examined the benefits of EPA + DHA instead of purified EPA formulations as compared to JELIS and REDUCE-IT, indicating that combining EPA and DHA may have contributed to the lack of significance in reducing CVD outcomes. EPA and DHA have different biological effects on our cardiovascular system as DHA is known to moderately increase LDL-C compared to EPA in patients with elevated TG levels [92][93][94], thereby contributing to the inconsistent findings [8]. The increased use of medications lowering CVD risks such as statins, beta-blockers, and anti-coagulants may have also diluted the potential benefits of n-3 PUFA [30].
As discussed in the "Cancer section", large RCTs showed little or no effect on cancer and cancer mortality risks. Findings from large-scale trials such as ASCEND, JELIS, ORI-GIN, and VITAL corroborated the null results of a recently published SRMA of RCTs by Hanson et al., [12]. Data surrounding the effects of n-3 PUFA on different cancers have been inconsistent and were mostly based on experimental models and epidemiological studies [95]. In line with CVD, the varying doses of n-3 PUFA, poor allocation concealment, lack of blinding, shorter follow-up time to detect cancer incidence, and the medications prescribed to the study participants may have contributed to the lack of effects on cancer and its related outcomes [12,45]. Moreover, most trials with cancer data were not originally designed to look at the effects of n-3 PUFA on cancer risk, but CVD risk, which may have further contributed to the inconsistency of the evidence [45]. Since cancer is defined as an umbrella of more than 100 types of cancer, future research should examine the effects of n-3 PUFA specific to each cancer type, without generalizing across all cancers in order to observe a true degree of benefit [96]. Although, most of the participants from the large-scale trials such as JELIS, ORIGIN, ASCEND, and VITAL were overall healthy, it is important to note that those participating in trials are generally more health conscious, which may have mitigated the effects of n-3 PUFA on cancer and its related outcomes.
In addition to influencing CVD and cancer outcomes, n-3 PUFAs are also known to influence parameters of type 2 diabetes such as insulin resistance (IR), fasting blood glucose (FBG), and HbA1c. Inconsistent findings in the diabetes literature may be attributed to a lack of observed changes in inflammatory responses, shorter follow-up time, smaller sample size, inadequate dosage of n-3 PUFA, open-label design, higher attrition rate, combined intervention, baseline plasma glucose levels, health status of the study participants, HOMA-R values, and lack of sensitive methods such as the use of euglycemic clamp to detect any changes in insulin sensitivity [52,[55][56][57]63]. Studies observing beneficial effects of n-3 PUFA on type 2 diabetes and its parameters also used combined interventions such as n-3 PUFA with plant sterol or vitamin D. Both plant sterol and vitamin D are known to prevent impaired glucose regulation (IGR) progression to type 2 diabetes and enhance the insulin-regulated glucose transporter type-4 (GLUT-4), respectively, while improving blood glucose levels and insulin resistance [61,62]. Therefore, the significant results from the combined intervention may have resulted in an overestimation of the true effects of n-3 PUFA due to the beneficial effects of plant sterols and vitamin D.
Conversely, null effects on FPG and HbA1c were observed after supplementing diets with 1.8 g of EPA daily for 6 months in the Japanese population. This null effect was due to the mean homeostasis model assessment ratio (HOMA-R) of 1.6, which was closer to the upper limit of normal HOMA-R values in the Japanese population, and as a result prevented any further reduction in FPG and HbA1c [57]. Sawada and colleagues also noted that those randomized in the EPA group had significantly lower plasma blood glucose levels than the placebo group, therefore adding to the lack of effect [57]. As Delpino et al. mentioned, 20% of their included studies in their SRMA were not double blind, which as a result may influence the behavior and responses of the participants to the treatment [52]. Additionally, study participants with various health conditions such as impaired fasting blood glucose, metabolic syndrome, vitamin D deficiency, polycystic ovarian syndrome, and hepatis may have led to the inconclusive findings on diabetes and its related parameters.
In line with the inconsistent findings related to n-3 PUFA research, the long-term consumption of n-3 PUFA has not shown a significant reduction on the incidence and progression of AMD. After a median follow-up of approximately 5 years, no significant reduction in AMD incidence and progression to advanced AMD were observed in the intervention group receiving n-3 PUFA in large-scale trials such as AREDS and VITAL [78,79]. These studies had a low attrition rate, large sample size, and a high level of adherence to the treatment regimen. The lack of benefit of n-3 PUFAs were attributed to the duration of the trial, the form of EPA and DHA used, as well as the EPA and DHA ratio since the ratio used was mostly designed for CVD related studies [78,79]. In the AREDS study, the complex secondary randomization design may have further made it more difficult to understand the role of EPA and DHA as they were combined with lutein and zeaxanthin, as well as the AREDS formulation, which consisted of vitamin C, vitamin E, beta carotene, zinc, and copper [78]. All AREDS participants took the usual AREDS formulation, leading us to believe that there may not have been a true placebo to evaluate the true effects of n-3 PUFA [79]. The results from AREDS may also not be generalizable since it was conducted among a highly educated and "well-nourished" group of individuals [78]. The VITAL trial also showed null effects on AMD incidence and progression, and it may be because of its low-risk population without any prior AMD and the "under ascertainment" of AMD, thus reducing the study power [79].

Strengths and Limitations
This review has a few strengths and limitations that should be addressed. First, this was a comprehensive review where the highest level evidence from n-3 PUFA RCTs across chronic diseases such as CVD, cancer, type 2 diabetes, and AMD were reviewed and critically analyzed. However, a potential limitation should also be considered. The totality of evidence was not considered since our focus was on high quality and large RCTs, especially for CVD, cancer, and AMD. Nevertheless, these RCTs provide important perspectives for future studies of any size.

Conclusions
In conclusion, the evidence from RCTs examining effects of n-3 PUFA on CVD, cancer, type 2 diabetes and its related parameters, and AMD was dependent on the types of trials, whether they were primary or secondary prevention, trial design, varying doses and forms of n-3 PUFA, duration of the trials, baseline characteristics of the study participants, ethnicity, geographical locations, health status of study population, the sample size, daily intake of n-3 PUFA, attrition rate, and participants' adherence to the treatment regimen. To understand the controversies and inconsistencies surrounding n-3 PUFA, we must therefore temper our expectations around RCTs and recognize that in general, n-3 PUFA is not a pharmaceutical drug. We also must recognize that n-3 PUFA nutrition trials are not poorly done but have limitations and that the evidence from these RCTs may be stronger than they really are. The findings from this review of RCTs and n-3 PUFA studies also suggest that there is a need for future research to distinguish between primary and secondary prevention, and to focus more on primary prevention of the aforementioned chronic diseases and determine whether a significant and beneficial effect of n-3 PUFA may be obtained. Primary prevention studies will not be conflicted by co-morbidities, the use of other prescription drugs or other significant confounders. Although the research surrounding n-3 PUFA and age-related chronic diseases are mostly focused on treatments with EPA and DHA combined or EPA alone, future trials of DHA monotherapy are needed to confirm the mechanistic function of DHA since EPA and DHA differ in their biological effects. Overall, the expectations from large n-3 PUFA RCTs have not yielded conclusive evidence, but merely reaffirmed the complexity of human based nutrition research and the need to consider the totality of all study designs and the uniqueness of many different population groups.